1. Field of the Invention
The present invention relates generally to the field of plant breeding and molecular biology. In particular, the invention relates to agronomically elite soybean varieties with increased beta-conglycinin content and materials for making such plants.
2. Description of Related Art
Glycinin and β-Conglycinin are two major storage proteins in soybeans, accounting for approximately 70% of total proteins or 40% of total seed weight. The glycinin (11s globulin) is composed of five different subunits, designated A1aB2, A2B1a, A1bB1b, A5A4B3, A3B4, respectively. Each subunit is composed of two polypeptides, one acidic and one basic, covalently linked through a disulfide bond. The two polypeptide chains result from post-translational cleavage of proglycinin precursors, a step that occurs after the precursor enters the protein bodies (Chrispeels et al., 1982). Five major genes have been identified to encode these polypeptide subunits. They are designated as Gy1, Gy2, Gy3, Gy4 and Gy5, respectively (Nielsen et al., 1997). In addition, a pseudogene, gy6, and minor gene, Gy7, were also reported (Beilinson et al., 2002). Genetic mapping of these genes has been reported by various groups (Diers et al., 1993, Chen and Shoemaker 1998, Beilinson et al., 2002). Gy1 and Gy2 were located 3 kb apart and mapped to linkage group N (Nielsen et al., 1989), Gy3 was mapped to linkage group L (Beilinson et al., 2002). Gy4 and Gy5 were mapped to linkage groups O and F, respectively. All of these genes were mapped using RFLP probes on Southern Blots.
β-conglycinin, on the other hand, is composed of α (˜67 kda), α′ (˜71 kDa) and β (˜50 kDa) subunits and each subunit is processed by co- and post-translational modifications (Ladin et al., 1987; Utsumi, 1992). The β-conglycinin subunits are encoded by the genes Cgy1, Cgy2 and Cgy3, respectively. Genetic analysis indicated that Cgy2 is tightly linked to Cgy3, whereas Cgy1 segregates independently of the other two. The β-conglycinin gene family contains at least 15 members divided into two major groups, which encode the 2.5 kb and 1.7 kb embryo mRNA, respectively (Harada et al., 1989).
Soybean plants with increased β-conglycinin levels and decreased glycinin levels would provide substantial benefit. One reason for this is that β-conglycinin is a soluble protein whereas glycinin is much less soluble. It has also been found that β-conglycinin, especially the α′ subunit, has significantly higher nutritional value and a positive impact on human health as compared to glycinin (Baba et al., 2004). A number of experiments using animal models have indicated that α′ subunit from soybean β-conglycinin could lower plasma triglycerides, and also increase LDL (“bad” cholesterol) removal from blood (Duranti et al., 2004, Moriyama et al., 2004, Adams et al., 2004, Nishi et al., 2003). Therefore, soybean varieties with an increased β-conglycinin content will have higher value than traditional varieties and will be suitable for use in nutrition drinks and other food products.
Interestingly, mutations in the glycinin genes have a direct impact on β-conglycinin content in soybean seeds. Mutant soybean plants with decreased glycinin content have increased β-conglycinin content. However, since multiple glycinin alleles are involved in glycinin subunit production, breeding plants with reduced expression from multiple Gy subunits has proved difficult since such plants have other attributes, such as low yield, excessive lodging and green seed that render them commercially nonviable. Previous methods for determining the inheritance of mutations resulting in decreased glycinin content did not enable high-throughput techniques required to select for these phenotypes while introducing agronomically superior characteristics. For example, previous assessment of Gy inheritance was dependent upon analysis of protein expression, which is costly, labor intensive and cannot track the inheritance of recessive mutations. The possibility of producing such plants regardless of labor was also unknown, due to additional complications such as linkage drag and epistasis associated with attempts to introgress a mutant Gy allele. The combination of alleles are also unpredictable with respect to the phenotype obtained. Thus, there is a longstanding but unfilled need in the art for agronomically elite soybean plants with reduced expression of multiple Gy protein subunits and methods for production of such plants.
Lipoxygenases are enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. Soybean seeds contain three lipoxygenase isozymes—lipoxygenases 1, 2, and 3. These isozymes contribute to the production of unpleasant flavors in soybean seeds. The unpleasant flavors are absent or less pronounced in seeds deficient in these isozymes, particularly those lacking lipoxygenase-2. Accordingly, soybean seeds lacking one or more lipoxygenase isozymes are desirable for use in making drink and food products. Genetic studies of Lipoxygenase 1, 2, and 3 deficient lines demonstrated that the absence of each was due to single recessive alleles—lx1, lx2, and lx3, respectively. The loci defined by lx1 and lx2 are closely linked and are not genetically linked to lx3 (Kitamura, 1984; Kitamura et al, 1985; Hajika et al., 1992; Hildebrand et al., 1982). The structural genes encoding Lipoxygenases 1, 2, and 3 have been cloned and designated Lox1, Lox2, and Lox3, respectively (Shibata et al., 1987; Shibata et al., 1988; Yenofsky et al., 1988).
Kunitz Trypsin inhibitor (KTI) is an antinutritional and allergenic factor in soybeans that interferes with digestion and absorption of proteins when present in a diet. Thus, soybean varieties with a KTI-null mutant trait have a higher commercial value than traditional varieties. Genetic and biochemical studies of KTI production in soybean lines have been carried out (e.g. de Moraes et al., 2006; Natarajan et al., 2006), and three related genes have been identified, with KTI3 encoding the predominant Kunitz Trypsin Inhibitor Protein in cultivated soybean genotypes (Natarajan et al., 2006). Some specific DNA markers associated with loss of KTI production in certain soybean lines have been reported (de Moraes et al., 2006).